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Adiponectin stimulates autophagy and reduces oxidative stress to enhance

insulin sensitivity during high fat diet feeding in mice

Ying Liu1, Rengasamy Palanivel1, Esther Rai1, Min Park1,

Tim V. Gabor1, Michael P. Scheid1, Aimin Xu2 & Gary Sweeney1

1Department of Biology, York University, Toronto, Canada

and 2State Key Laboratory of Pharmaceutical Biotechnology, and Department of Medicine, the University of Hong Kong

Corresponding author Dr. Gary Sweeney Department of Biology York University 4700 Keele Street Toronto, ON, M3J 1P3 Canada Tel: 416-736-2100 Fax: 416-736-5698 Email: [email protected]

Keywords: Adiponectin, autophagy, oxidative stress, insulin sensitivity, skeletal muscle

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Diabetes Publish Ahead of Print, published online July 28, 2014

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Abstract

Numerous studies have characterized the anti-diabetic effects of adiponectin yet the

precise cellular mechanisms in skeletal muscle, in particular changes in autophagy,

require further clarification. In the current study we used high fat diet (HFD) to induce

obesity and insulin resistance in wild type (wt) or adiponectin knockout (Ad-KO) mice ±

adiponectin replenishment. Temporal analysis of glucose tolerance and insulin sensitivity

using hyperinsulinemic-euglycemic clamp and muscle IRS and Akt phosphorylation

demonstrated exaggerated and more rapid HFD-induced insulin resistance in skeletal

muscle of Ad-KO mice. SOD activity, GSH/GSSG ratio and lipid peroxidation indicated

that HFD-induced oxidative stress was corrected by adiponectin and gene array analysis

implicated several antioxidant enzymes, including Gpxs, Prdx, Sod and Nox4 in

mediating this effect. Adiponectin also attenuated palmitate-induced ROS production in

cultured myotubes and improved insulin-stimulated glucose uptake in primary muscle

cells. Increased LC3-II and decreased p62 expression suggested that HFD induced

autophagy in muscle of wt mice, however these changes were not observed in Ad-KO

mice. Replenishing adiponectin in Ad-KO mice increased LC3-II and Beclin1 and

decreased p62 protein levels, inducec FGF-21 expression and corrected HFD-induced

decreases in LC3, beclin1 and ULK1 gene expression. In vitro studies examining

changes in phospho-ULK1(Ser555), LC3-II and lysosomal enzyme activity confirmed

that adiponectin directly induced autophagic flux in cultured muscle cells in an AMPK-

dependent manner. We overexpressed an inactive mutant of Atg5 to create an

autophagy-deficient cell model and, together with pharmacological inhibition of

autophagy, demonstrated reduced insulin sensitivity under these conditions. In

summary, adiponectin stimulated skeletal muscle autophagy and antioxidant potential to

reduce insulin resistance caused by HFD.

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Introduction

Adiponectin normally circulates abundantly in the concentration range of 2-

20ug/ml and decreased plasma adiponectin, in particular the high molecular weight

(HMW) form, has been found in patients with obesity and type 2 diabetes (1). Extensive

studies have shown that adiponectin exerts beneficial anti-diabetic actions via direct

metabolic and insulin-sensitizing effects in various tissues (2). Skeletal muscle is the

major site for glucose disposal and maintenance of insulin sensitivity is critical for

optimal glucose homeostasis. Generation of reactive oxygen species (ROS) and the

resulting oxidative stress, mitochondrial dysfunction and accumulation of triglyceride and

lipotoxic metabolites have all been shown to contribute to insulin resistance (3; 4).

Transgenic mice overexpressing adiponectin show improved insulin sensitivity and

mitochondrial function (5; 6) while Ad-KO mice are more susceptible to HFD-induced

insulin resistance.

In response to cellular stressors, increased levels of autophagy permit cells to

efficiently adapt by altering protein catabolism, however autophagy is viewed as a

double edged sword, with too much or too little and the temporal nature of the process

determining cellular consequences (7; 8). Several studies have recently begun to

establish the importance of autophagy in skeletal muscle metabolism. In autophagy-

deficient mice with skeletal muscle-specific deletion of Atg7 the induction of Fgf21

expression in muscle mediated peripheral effects leading to protection from diet-induced

obesity and insulin resistance (9). Another mouse model of stimulus-deficient autophagy,

the BCL2 AAA mice which contain knock-in mutations in BCL2 phosphorylation sites

(Thr69Ala, Ser70Ala and Ser84Ala) that prevent autophagy activation, showed altered

glucose metabolism during acute exercise, as well as impaired chronic exercise-

mediated protection against high-fat-diet-induced glucose intolerance (10). Activation of

autophagy in skeletal muscle has been reported by others in response to exercise and

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caloric restriction (11-14). The ability to induce autophagy has been shown to deteriorate

with aging in skeletal muscle (15). Thus, recent literature suggests that induction of

skeletal muscle autophagy by various stimuli may give rise to beneficial metabolic

effects. Numerous studies have also established the importance of crosstalk between

autophagy and oxidative stress (16).

It is clear that adiponectin exerts beneficial metabolic effects by direct actions

and enhancing insulin sensitivity (2), however the underlying molecular mechanisms are

incompletely understood. We used Ad-KO mice fed HFD for 2, 4 and 6 weeks with and

without adiponectin replenishment to examine corrective effects of adiponectin in this

model. We examined changes in oxidative stress and underlying mechanisms and also

established that adiponectin directly stimulates autophagy in skeletal muscle. These

studies provide new insight into skeletal muscle actions of adiponectin which contribute

to the beneficial anti-diabetic effects of this adipokine.

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Materials and Methods

Reagents and Antibodies

3H-2-Deoxy-glucose was purchased from PerkinElmer (Ontario, Canada). Insulin

(Humulin R) was purchased from Eli Lilly (Toronto, Canada). TRIzol reagent was from

Invitrogen Life Technologies (Burlington, ON). Polyclonal phosphospecific antibodies to

Akt (Thr308, Ser473) and ULK1 (Ser555), LC3B, Beclin1, GAPDH and horseradish

peroxidase (HRP)-conjugated anti-rabbit-IgG were from Cell Signaling Technology

(Beverly, MA) while polyclonal phosphospecific antibody to IRS1 (Y612) was from Life

Technologies (Burlington, ON), p62 antibody from BD Biosciences (Mississauga, ON)

and FGF21 antibody from Antibody Immunoassay Services (Hong Kong). Polyvinylidene

difluoride (PVDF) membrane was from Bio-Rad (Burlington, ON) and

chemiluminescence reagent plus from PerkinElmer (Boston, MA). AMEM and FBS were

purchased from GIBCO®, Invitrogen life technology (Burlington, ON). All other reagents

and chemicals used were of the highest purity available.

Experimental animals, glucose tolerance test (GTT) and hyperinsulinemic euglycemic

clamp

Animal facilities met the guidelines of the Canadian Council on Animal Care, and the

protocols were approved by the Animal Care Committee of York University. Animals

were fed either regular chow diet or 60% HF diet as we described (22). HF diet AdKO

animals received either saline or adiponectin (3µg/g body weight) twice daily for 1wk and

2wks for the 2wks diet and 6wks diet groups, respectively, via intraperitoneal injection.

GTT and clamp studies were performed as we described (22).

Preparation of muscle homogenates, cell lysates and Western blotting

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Stably transfected L6 cells were serum starved 4 hr or incubated with or without

bafilomycin (200nM, 24hr), chloroquine (100µM, 24hr), compound C (10µM, 1hr)

followed by insulin stimulation (10, 100 nM, 5 min) or adiponectin (5µg/ml, 30 and 60

min) treatment. All tissue and cell samples were prepared as we described before (22)

and primary antibodies used at 1:1000 dilution. Membranes were then washed four

times in 1x wash buffer for 15 min each at room temperature and incubated with

appropriate HRP-coupled secondary antibody (1:10,000) for 1 h. Membranes were

washed five times in 1x wash buffer for 10 min each and proteins visualized using

enhanced chemiluminescence. Non-denaturing, non-reducing conditions were applied to

allow analysis of different forms of adiponectin (HMW>250KDa, MMW=180KDa and

LMW=90KDa) with in-house polyclonal anti-adiponectin antibody in the concentration of

1ug/ml and anti-rabbit as secondary antibody. Quantitation of each specific protein band

was then determined via densitometric scanning and correction for the respective

loading control.

Superoxide dismutase (SOD) activity assay

Tissue specific SOD activity was assessed with a colorimetric kit “Superoxide Dismutase

Activity Assay Kit” purchased from Biovision (California, USA). Gastrocnemius skeletal

muscle samples were powderized in liquid N2 and protein extracted according to

manufacturer’s instruction. Approximately 10µg protein from each sample was used to

measure SOD activity under OD450nm by a microplate photometric reader. Data

represented in the results section were normalized for protein concentration loaded for

each sample.

Glutathione (GSH) assay

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Tissue specific total and reduced GSH level were analyzed with a “ApoGSHTM

Glutathione Colorimetric Detection Kit” from Biovision (California, USA). Tibialis Anterior

(TA) skeletal muscle samples were crushed into powder in liquid N2 and protein

extracted according to manufacturer’s instruction. Same volume of lysate for each

sample was used to determine total and reduced GSH level and assay read at 412nm by

a microplate photometric reader. Data represented in the results section were

normalized by protein concentration loaded for each sample.

TBARS assay

Tissue specific lipid peroxidation level was analyzed with a “Thiobarbituric Acid Reactive

Substances (TBARS) Assay Kit” from Cayman Chemical (Michigan, USA). Tibialis

Anterior (TA) skeletal muscle samples were crushed into powder in liquid N2 and protein

extracted according to manufacturer’s instruction. In total 600ug protein from each tissue

sample lysate was used to determine lipid peroxidation level and assay read at 540nm

by a microplate photometric reader. Data represented in the results section were

normalized by protein concentration loaded for each sample.

Oxidative stress-related and autophagy-related gene expression analysis.

Gastrocnemius skeletal muscle samples collected from animals were powderized in

liquid N2 and total RNA extracted with TRIzol reagent (Invitrogen Life Technologies,

Burlington, ON) and cleaned up by RNEasy mini-ki (Qiagen, Toronto, Ontario). cDNAs

were synthesized by reverse transcription with 1µg total RNA by using RT² First-Stand

cDNA Synthesis Kit (Qiagen). A mixture of cDNAs and RT2 qPCR Master Mix was

aliquoted to 96-well plates pre-coated with primers encoding different genes involved in

the regulation of oxidative stress pathway (Qiagen). The plate was loaded onto a qPCR

machine and real time PCR program was set according to the company’s instruction.

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Results collected from each well after qPCR were adjusted by several housekeeping

genes including GAPDH, beta-actin before analyzing. For analysis of autophagy-related

gene expression, RNA (0.2 µg) was reverse-transcribed in a 20-µl reaction volume using

specific primers (Invitrogen) listed in supplementary table 1 and GoScript reverse

transcriptase according to the manufacturer's instructions (Promega). RT-PCR was

performed with KAPA Polymerase Chain Reaction Master Mix (KAPA Biosystem Inc,

Wilmington, MA, USA) and SYTO® 9 Green Fluorescent Nucleic Acid Stain (Life

Technologies) under standard thermocycling conditions (2 min at 95°C and followed by

40 cycles of 5 s each at 95, 60, and 72°C). Relative expression levels of genes,

normalized using beta-actin as a housekeeping gene, were calculated using the

comparative critical threshold method.

Immunofluorescent analysis of endogenous LC3

Analysis of LC3 cellular localization was performed by culturing cells on glass cover slips

and treating with adiponectin for 1hr and 2hrs. Thereafter, the media was aspirated and

the cells were washed (3X) in phosphate-buffered saline (PBS) at room temperature and

then fixed for 20 min in 4% paraformaldehyde (PFA) at room temperature, then further

washed (3X for 5 min) with PBS. Cells were permeabilized with 0.1% Triton X-100 for 3

minutes and blocked with blocking buffer (3% BSA) for 30 minutes. Following fixation,

permeabilization and blocking, the cells were washed once in PBS and then sequentially

stained with LC3 primary antibody (1:1000) overnight at 40C then secondary antibody

conjugated with Alexa Fluor 488 (1h at room temperature). Cells were then washed with

PBS before incubation with 2 µg/ml 4,6-diamidino-2-phenylindole (DAPI, Roche

Diagnostics) in PBS, 20 min at room temperature. The samples were washed with PBS

(4X) and finally mounted with Dako fluorescence mounting medium (Dako North

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America Inc, CA) and observed with an Olympus confocal microscope equipped with a

60X objective.

Cell culture using L6 and primary skeletal muscle cells.

We used the L6 skeletal muscle cell line grown as previously described (17) for some in

vitro studies as indicated in figure legends. To prepare primary skeletal muscle cells we

also isolated muscle strips from mouse hind leg and cut them into small pieces which

were then incubated in 3ml dipase/collagenous solution (0.1 type II collagenase + 0.05%

dipase in serum free Ham’s F12 media) for 30mins in 37ºC water bath with agitation

(~100rpm). Digested solutions were then filtered through a 100um cell filter, and only

flow through was collected and centrifuged at room temperature for 7mins at 1600rpm.

The cell pellet was then resuspended in growth media (10% FBS, 0.5% antibiotics and

antimycotics, 5ng/ml rhFGF in Ham’s F12) and plated onto a 35mm dish overnight. The

supernatant which contained non-adherent cells were then seeded on a laminin-coated

35 mm dish. These cells were differentiated into myotubes in Ham’s F12 media

containing 2% horse serum for 5-7 days and experiments were performed using fully

differentiated myotubes.

Generation of L6 cell line with stable overexpression of tandem RFP/GFP-LC3 (tfLC3) or

ATG5-K130R by retroviral infection

We used the L6 skeletal muscle cell line for in vitro studies. After identifying relevant and

unique restriction sites matching with the retroviral cloning vector pQCXIP, and

RFP/GFP-LC3 target vector, ptfLC3 (Addgene), the retroviral vector expressing the gene

of interest, tfLC3, and all other essential elements for retroviral integration and

expression was successfully generated and purified. We also subcloned the target RFP-

ATG5-K130R sequence from the vector pmCherry-ATG5-K130R (Addgene) into the

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vector pQCXIP. In both cases, the viral vector was then transfected into HEK-derived

packaging cell line, EcoPack 2-293 (Clontech), expressing the MMLV Gag, Pol and Env

proteins. The culture medium containing the virus was collected 48 hours post

transfection. 100 ul of the collected supernatant, or viral soup, was directly applied to L6

cells in presence of polybrene (4ug/ml) in 10 cm culture dish on the next day after being

seeded. Cells were incubated with the virus for 24 hours in the incubator, and the

medium was replaced with fresh growth medium containing the selection antibiotic,

puromycin (2ug/ml) (Sigma Aldrich). The pool of cells resistant to the antibiotic was

selected, and the stable overexpression of the target gene was verified by detecting the

expression of GFP using FACS Calibur flow cytometry (BD Bioscience).

Analysis of autophagic flux using tandem RFP/GFP-LC3

L6 cells stably transfected with tfLC3 were seeded on glass cover slips and cells were

then maintained in culture medium until reaching 70–80% confluence then treated with

adiponectin (5ug/ml up to 24hr). Cells were then washed with PBS (phosphate-buffered

saline) and fixed with 4% paraformaldehyde for 20 min, followed by permeabilization

with 0.1% Triton X-100 for 3 minutes. Cells were blocked with PBS containing 3%BSA

for 30 minutes at room temperature. After washing three times with PBS, coverslips

were mounted with Dako fluorescence mounting medium (Dako North America Inc, CA),

and GFP and RFP fluorescence detected by confocal microscopy.

Analysis of cathepsin B activity using MagicRed

L6 cells were cultured on glass coverslips and treated with adiponectin for 1h and 2hr

then lysosomal activity determined with Magic Red Cathepsin B kit (Immunochemistry

Technologies, Bloomington, MN) according to the manufacturer's instruction. Nuclei

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were counterstained with DAPI for 20 min. After washing (4X) with PBS, cells were

mounted with Dako fluorescence mounting medium and images were observed with an

Olympus confocal microscope equipped with a 60X objective.

2-Deoxy-glucose uptake

L6 myoblasts stably overexpressing GLUT4-myc (17) were incubated with media

contaiing 0.5% FBS with or without bafilomycin (200nM, 24hr) or chloroquine (100µM,

24hr) followed by insulin (10 and100 nM, 20 min). After treatments, glucose uptake was

determined as previously described (17).

Statistical analysis

All data were analyzed by using Graphpad Prism 5 and presented as means ± S.E.M.

One-way or two-way ANOVA was performed where appropriate and differences were

considered statistically significant at P<0.05.

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Results

We first validated the model used here to study the mechanisms via which adiponectin

improved insulin sensitivity. Temporal analysis after 2, 4 and 6 weeks of HFD of area

under curve upon glucose tolerance test (Fig 1A), plasma insulin (Fig 1B), plasma

glucose (Fig 1C) and HOMA-IR (Fig 1D) indicated an exaggerated and more rapid

development of insulin insensitivity in Ad-KO mice subjected to HFD when compared

with wt mice. This was reflected in significantly reduced insulin-stimulated

phosphorylation of IRS(Y612) and Akt(S473) in skeletal muscle of Ad-KO mice at 4 and

6 weeks, yet only after 6 weeks in wt mice (Figs 1E&F). Original Western blot data is

shown for all insulin signaling analysis in supplementary figure 1. Detailed investigation

of insulin sensitivity using hyperinsulinemic-euglycemic clamp in these mice

demonstrated impaired glucose homeostasis in Ad-KO mice after only two weeks HFD.

This was evident from decreased glucose infusion rate (Fig 2A) and insulin altered

glucose appearance rate (Fig 2B&E), despite an increased glucose disappearance rate

(Fig 2C&E). After 2wks high fat diet, but not after 6 weeks (22), we observed an

elevation in insulin stimulated glucose uptake in skeletal muscle, indicating an important

initial compensatory role of skeletal muscle in maintaining whole body glucose

homeostasis in the Ad-KO mouse. There was no difference observed in the glycolytic

rate in Ad-KO mice fed either chow or HFD for 2wks (Fig 2D).

We next examined changes in oxidative stress and observed a rapid significant

decrease in SOD activity after 2 weeks of HFD in Ad-KO, but not wt, mice (Fig 3A). After

4 weeks, HFD-induced a decrease in SOD activity in both groups of mice. Interestingly,

this decreased SOD activity was transient and recovered in both wt and HFD mice after

6 weeks HFD (Fig 3A). Under chow fed conditions, Ad-KO mice had a lower GSH/GSSG

ratio than wt mice (Fig 3B) and there was an increase in GSH/GSSG ratio in muscle of

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Ad-KO mice after 4 and 6 weeks of HFD (Fig 3B). Wt mice showed an apparent

decrease after 2 weeks and a significant decrease in GSH/GSSG ratio after 4 weeks of

HFD (Fig 3B). After replenishing normal circulating levels of adiponectin to Ad-KO mice

we observed that the HFD-induced decrease in SOD activity was corrected (Fig 3C).

Furthermore, analysis of lipid peroxidation using TBARS assay confirmed an elevated

level of oxidative stress in muscle of Ad-KO mice after 2 weeks of HFD and this was

corrected in mice which received adiponectin replenishment (Fig 3D). Analysis of ROS

generation in response to palmitate in vitro using L6 myotubes confirmed adiponectin’s

effect on preventing free fatty acid induced oxidative stress (Fig 3E). We measured

glucose uptake in primary skeletal muscle cells to determine changes in insulin

sensitivity and found that adiponectin enhanced sensitivity of cells to submaximal (10nM)

insulin concentration and that palmitate-induced insulin resistance was alleviated in the

presence of adiponectin (Fig 3F).

To further investigate potential mechanisms underlying these changes we performed

gene array analysis of those implicated in regulation of oxidative stress. Within a total of

84 genes which were analyzed, high fat diet altered the expression of genes involved in

the regulation of not only glutathione peroxidase, peroxiredoxin, superoxide dismutase,

superoxide metabolism but also oxidative stress responsive and oxygen transporter

genes (Fig 4A). Adiponectin supplementation effectively reversed HFD-induced changes

in expression of several of the main anti-oxidant genes: Gpx1&3, SOD1&2, Prdx1,3&4,

Ptgs 1&2 (Fig 4B). HFD also induced the reactive oxygen species production gene Nox4

and a direct upregulation of the copper chaperone for superoxide dismutase (Ccs) by

adiponectin was also observed (Fig 4B). A heat map showing specific information on the

full range of genes tested and changes therein is shown in supplementary figure 2.

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We investigated temporal changes in skeletal muscle autophagy in these mice using

well established markers. HFD feeding for 6 weeks significantly increased levels of LC3-

II in wt skeletal muscle, but this was not observed in Ad-KO mice (Fig 5A). Original

Western blot data is shown for all autophagy analysis in supplementary figures 3 and 4.

We found that replenishing adiponectin to Ad-KO mice restored the higher level of LC3-II

expression (Fig 5B). A decrease in p62 levels was observed at all time points in wt

mouse muscle, however accumulation of p62 was evident in Ad-KO mouse muscle (Fig

5C), the latter being reduced by adiponectin supplementation (Fig 5D). The enhanced

LC3-II levels observed after 6 weeks of HFD in wt mice correlated with enhanced

expression of beclin1 (Fig 5E). Beclin1 expression did not change in Ad-KO mice,

however adiponectin did induce beclin1 expression in muscle of these mice (Fig 5E).

Skeletal muscle FGF-21 content was enhanced after 2, 4 and 6 weeks of HFD only in wt

mice (Fig 5G), however replenishing adiponectin in Ad-KO mice led to increased FGF-21

expression (Fig 5H). We also analyzed changes in expression of genes which play a key

role at various stages of autophagic flux. Our data indicated that HFD decreased

expression of several autophagy-related genes and three of these, beclin1, LC3 and

ULK1, were corrected by adiponectin treatment (Fig 5I).

We then examined changes in autophagy in cultured skeletal muscle cells treated with

or without adiponectin. Conversion of LC3-I to LC3-II is the most widely used marker for

autophagosomes and when this was assessed by Western blotting (Fig 6A) we found

increased LC3-II levels after adiponectin treatment. We hypothesized that the

mechanism via which adiponectin stimulated autophagy was via AMPK and after

inhibition of AMPK using compound C, adiponectin stimulated LC3-II formation and

phosphorylation of ULK1 on Ser555 was attenuated (Fig 6A). Autophagosome formation

in response to adiponectin was also confirmed by punctate appearance of endogenous

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LC3 upon immunofluorescent detection (Fig 6B). To more accurately examine

autophagic flux we generated skeletal muscle cells stably overexpressing RFP/GFP-

LC3. This approach is advantageous in that it allows analysis of autophagic flux since

GFP, but not RFP, is sensitive to quenching by the acidic environment of the

autophagolysosome. We observed that adiponectin initially stimulated punctate

appearance of fluorescent LC3 while comparison of merged images showing maintained

red and decreased green fluorescence indicated completion of autophagic flux in

response to adiponectin (Fig 6C). Lysosomal enzyme activity, measured using

MagicRed, was increased in response to adiponectin (Fig 6D). We used bafilomycin or

chloroquine to inhibit autophagic flux and demonstrated that this led to decreased

insulin-stimulated glucose uptake (Fig 7A). Similarly, insulin-stimulated phosphorylation

of IRS1 (Tyr612), Akt (Thr308) and Akt (Ser473) was attenuated under these conditions

as seen in the representative Western blots (Fig 7B) and quantitative analysis (Fig 7C-

E). We also generated L6 skeletal muscle cells stably overexpressing the dominant-

negative inhibitor of autophagy (Atg5K130R). In these autophagy-deficient cells we also

observed a reduction in insulin stimulated phosphorylation of IRS1 and Akt as seen in

the representative Western blots (Fig 7F) and quantitative analysis (Fig 7G-I).

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Discussion

Numerous studies have demonstrated that adiponectin improves insulin sensitivity and

alleviates metabolic dysfunction in skeletal muscle (2; 18). Various mechanisms via

which adiponectin acts have been established, yet further detailed investigation and

uncovering of novel aspects is needed. Here, we used Wt and Ad-KO (± adiponectin

replenishment) mice fed chow or HFD. We previously validated that HFD-fed Ad-KO

mice developed skeletal muscle insulin resistance and glucose intolerance and that

replenishment of adiponectin corrected these defects (19). We also conducted a

metabolomic profiling analysis and found that adiponectin alleviated HFD-induced

changes in metabolites such as several diacylglycerol species, branched-chain amino

acids and various lysolipids (19). Here, an important goal of our study was to analyze

temporal changes (at 2,4,6 weeks) in glucose homeostasis (glucose tolerance test and

hyperinsulinemic-euglycemic clamp) and skelatal muscle insulin sensitivity to confirm an

exaggerated and more rapid effect in Ad-KO mice which was alleviated by administering

recombinant adiponectin.

Oxidative stress is well established as a causative factor in the development of skeletal

muscle insulin resistance and mitochondrial dysfunction and one which can be modified

by adiponectin (3; 4; 18; 20). One conclusion from our current study is that the ability of

adiponectin to correct HFD induced reductions in antioxidant gene expression is one of

the mechanisms via which adiponectin exerts its insulin sensitizing effect in skeletal

muscle and improves peripheral glucose homeostasis. Superoxide dismutase, catalase

and glutathione peroxidase are major anti-oxidative enzymes with which cells are

equipped to fight damage caused by ROS (21; 22). The increased expression of these

anti-oxidative enzymes was indeed correlated with measures of oxidative stress, such as

GSH/GSSG ratio, SOD activity and TBARS assay to assess lipid peroxidation. One

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additional interesting observation we made was that Ad-KO mice had higher initial

oxidative stress level compared to wt mice. The elimination of ROS can also be

catalyzed by nitric oxide synthase (Nos2), and we observed that expression of this

enzyme was reduced by HFD and induced by adiponectin. Other studies have

demonstrated that adiponectin reduced oxidative stress by down regulating NADPH

oxidase via inactivating the phosphorylation of P47phox, the regulatory subunit of NADPH

oxidase (23). Many studies have now established an important crosstalk between

oxidative stress and autophagy (24) although the significance in terms in skeletal muscle

metabolism (9-12; 14; 15) requires further investigation.

An important and novel focus of our study, therefore, was the analysis of skeletal muscle

autophagy (9-12; 14; 15). First of all, increased LC3-II and decreased p62 levels

indicated that HFD induced autophagy in skeletal muscle (25). The increased induction

of autophagy may be at least partly due to an increase in beclin-1, which has a well

characterized role in the induction of autophagosome formation (25). To date there have

been no studies documenting regulation of skeletal muscle autophagy by adiponectin,

however in recent months direct regulation of autophagy by adiponectin has been

shown in liver and macrophages (26; 27). Importantly, we found that the induction of

skeletal muscle autophagy observed in Wt mice was not apparent in Ad-KO mice.

However, replenishing adiponectin to the circulation of HFD-fed Ad-KO mice restored the

increase in LC3-II formation and reduced accumulation of p62. Based on our data and

recent studies which have begun to document the association between skeletal muscle

autophagy and metabolism (9; 10; 28; 29) we propose an important role of adiponectin

in the ability of skeletal muscle to elevate autophagy in response to HFD.

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To further investigate this observation we then examined whether adiponectin directly

induced autophagy in skeletal muscle cells. We measured autophagic flux using a

combination of approaches to determine autophagosome formation and

autophagolysosomal formation and activity (25), all of which demonstrated that

adiponectin directly stimulated autophagic flux. Our data on stimulation of autophagy by

adiponectin adds a novel perspective to the growing body of work documenting the

relationship of skeletal muscle autophagy with insulin sensitivity and metabolism. For

example, exercise stimulated skeletal muscle autophagy and autophagy-deficient mice

also exhibited altered glucose metabolism during acute exercise, as well as impaired

chronic exercise-mediated protection against HFD-induced glucose intolerance (10).

Activation of autophagy in skeletal muscle has been reported by others in response to

exercise and caloric restriction (11-14). Furthermore, starvation-induced autophagic flux

was greater in glycolytic versus highly oxidative muscle and this was related to AMPK

and mTOR activities which are both important determinants of autophagy (11; 13; 30;

31). The regulatory events required to induce autophagy were attenuated with aging in

skeletal muscle (15). We investigated the mechanistic role of AMPK in mediating the

stimulation of autophagic flux in skeletal muscle cells in response to adiponectin and

observed that the well established target of AMPK in inducing autophagy, ULK1

phosphorylation on Ser555, was directly increased by adiponectin. Furthermore,

stimulation of both LC3-II levels and phospho-ULK1 by adiponectin were attenuated

upon inhibition of AMPK, confirming an important role of AMPK signaling (26; 32). Thus,

both our current data and recent literature suggest that induction of skeletal muscle

autophagy by various stimuli gives rise to beneficial metabolic effects.

Perhaps the most striking observation in recent literature was that autophagy-deficient

mice with skeletal muscle specific deletion of Atg7 were protected from diet-induced

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obesity and insulin resistance (9). Mechanistically, in this model mitochondrial

dysfunction was overcome by autophagy-dependent FGF-21 expression in skeletal

muscle after 13 weeks of HFD. This FGF-21 acted as a myokine to mediate peripheral

effects leading to protection from diet-induced obesity and insulin resistance (9). We

investigated FGF-21 expression in skeletal muscle of HFD-fed Wt and Ad-KO mice and

our data indicated that only skeletal muscle from Wt, but not Ad-KO mice, had elevated

FGF-21 content. This suggests that HFD-induced autophagy and FGF-21 production

were dependent on adiponectin and we also observed that adiponectin directly induced

FGF-21 expression in cultured skeletal muscle cells. Interestingly, there are two recently

published reports that beneficial metabolic effects of FGF-21 are mediated via

adiponectin action (33; 34). Thus, conceivably, adiponectin may act as a front- and back-

end master regulator of FGF-21 physiology. The reciprocal interactions between

autophagy and oxidative stress (11) should also be further investigated, for example

using autophagy-deficient animal models.

In summary, this work significantly extends our understanding of mechanisms via which

adiponectin alleviates HFD-induced insulin resistance and metabolic dysfunction in

skeletal muscle. In particular, we document that HFD-induced obesity elicits increased

skeletal muscle autophagy and for the first time show the facilitatory role of adiponectin

in this process. We propose that adiponectin stimulates autophagic flux in skeletal

muscle, especially under pathological conditions, and that this represents an important

mechanistic component of its beneficial metabolic effects. Nevertheless, other cellular

events such as oxidative stress precede changes in autophagy and autophagy itself may

still be viewed as a doubled edged sword whereby too much or too little and the

temporal nature of the process can determine distinct cellular consequences (7; 8).

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Acknowledgements

This work was supported by an operating grant to GS from Canadian Institutes of Health

Research. GS also acknowledges Career Investigator support from Heart & Stroke

Foundation of Ontario. GS is guarantor of this article. No conflict of interest is declared

by any authors in this study. Author contributions: YL performed the majority of

experimental work, researched the work, helped in planning protocols and experiments

and with writing of manuscript. PR, ER, MP & TVG all conducted experimental work

included in the figures. MPS contributed to design of experimental work. AX contributed

to planning of study, analysis of data and editing of manuscript. GS designed the project,

supervised the experimental work, wrote the manuscript and provided funding.

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References

1. Liu Y, Retnakaran R, Hanley A, Tungtrongchitr R, Shaw C, Sweeney G: Total and

high molecular weight but not trimeric or hexameric forms of adiponectin correlate with

markers of the metabolic syndrome and liver injury in Thai subjects. J Clin Endocrinol

Metab 2007;92:4313-4318

2. Turer AT, Scherer PE: Adiponectin: mechanistic insights and clinical implications.

Diabetologia 2012;55:2319-2326

3. Newsholme P, Gaudel C, Krause M: Mitochondria and diabetes. An intriguing

pathogenetic role. Adv Exp Med Biol 2012;942:235-247

4. Samuel VT, Shulman GI: Mechanisms for insulin resistance: common threads and

missing links. Cell 2012;148:852-871

5. Combs TP, Pajvani UB, Berg AH, Lin Y, Jelicks LA, Laplante M, Nawrocki AR, Rajala

MW, Parlow AF, Cheeseboro L, Ding YY, Russell RG, Lindemann D, Hartley A, Baker

GR, Obici S, Deshaies Y, Ludgate M, Rossetti L, Scherer PE: A transgenic mouse with a

deletion in the collagenous domain of adiponectin displays elevated circulating

adiponectin and improved insulin sensitivity. Endocrinology 2004;145:367-383

6. Ge Q, Rycken L, Noel L, Maury E, Brichard SM: Adipokines identified as new

downstream targets for adiponectin: lessons from adiponectin-overexpressing or

deficient-mice. Am J Physiol Endocrinol Metab 2011;

7. Gurusamy N, Das DK: Is autophagy a double-edged sword for the heart? Acta Physiol

Hung 2009;96:267-276

8. Kubisch J, Turei D, Foldvari-Nagy L, Dunai ZA, Zsakai L, Varga M, Vellai T, Csermely

P, Korcsmaros T: Complex regulation of autophagy in cancer - Integrated approaches to

discover the networks that hold a double-edged sword. Semin Cancer Biol 2013;23:252-

261

9. Kim KH, Jeong YT, Oh H, Kim SH, Cho JM, Kim YN, Kim SS, Kim do H, Hur KY, Kim

HK, Ko T, Han J, Kim HL, Kim J, Back SH, Komatsu M, Chen H, Chan DC, Konishi M,

Itoh N, Choi CS, Lee MS: Autophagy deficiency leads to protection from obesity and

insulin resistance by inducing Fgf21 as a mitokine. Nat Med 2013;19:83-92

10. He C, Bassik MC, Moresi V, Sun K, Wei Y, Zou Z, An Z, Loh J, Fisher J, Sun Q,

Korsmeyer S, Packer M, May HI, Hill JA, Virgin HW, Gilpin C, Xiao G, Bassel-Duby R,

Scherer PE, Levine B: Exercise-induced BCL2-regulated autophagy is required for

muscle glucose homeostasis. Nature 2012;481:511-515

of 42 Diabetes

22

11. Mofarrahi M, Guo Y, Haspel JA, Choi AM, Davis EC, Gouspillou G, Hepple RT,

Godin R, Burelle Y, Hussain SN: Autophagic flux and oxidative capacity of skeletal

muscles during acute starvation. Autophagy 2013;9:1604-1620

12. Lira VA, Okutsu M, Zhang M, Greene NP, Laker RC, Breen DS, Hoehn KL, Yan Z:

Autophagy is required for exercise training-induced skeletal muscle adaptation and

improvement of physical performance. FASEB J 2013;27:4184-4193

13. Cui M, Yu H, Wang J, Gao J, Li J: Chronic caloric restriction and exercise improve

metabolic conditions of dietary-induced obese mice in autophagy correlated manner

without involving AMPK. J Diabetes Res 2013;2013:852754

14. Jamart C, Naslain D, Gilson H, Francaux M: Higher activation of autophagy in

skeletal muscle of mice during endurance exercise in the fasted state. Am J Physiol

Endocrinol Metab 2013;305:E964-974

15. Kim YA, Kim YS, Oh SL, Kim HJ, Song W: Autophagic response to exercise training

in skeletal muscle with age. J Physiol Biochem 2013;69:697-705

16. Wang Y, Li YB, Yin JJ, Wang Y, Zhu LB, Xie GY, Pan SH: Autophagy regulates

inflammation following oxidative injury in diabetes. Autophagy 2013;9:272-277

17. Ceddia RB, Somwar R, Maida A, Fang X, Bikopoulos G, Sweeney G: Globular

adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen

synthesis in rat skeletal muscle cells. Diabetologia 2005;48:132-139

18. Liu Y, Sweeney G: Adiponectin action in skeletal muscle Best Practice & Research

Clinical Endocrinology & Metabolism 2013;in press

19. Liu Y, Turdi S, Park T, Morris NJ, Deshaies Y, Xu A, Sweeney G: Adiponectin

corrects high-fat diet-induced disturbances in muscle metabolomic profile and whole-

body glucose homeostasis. Diabetes 2013;62:743-752

20. Matsuda M, Shimomura I: Roles of adiponectin and oxidative stress in obesity-

associated metabolic and cardiovascular diseases. Rev Endocr Metab Disord 2013;

21. Forstermann U: Nitric oxide and oxidative stress in vascular disease. Pflugers Arch

2010;459:923-939

22. Roberts CK, Barnard RJ, Sindhu RK, Jurczak M, Ehdaie A, Vaziri ND: Oxidative

stress and dysregulation of NAD(P)H oxidase and antioxidant enzymes in diet-induced

metabolic syndrome. Metabolism 2006;55:928-934

23. Carnevale R, Pignatelli P, Di Santo S, Bartimoccia S, Sanguigni V, Napoleone L,

Tanzilli G, Basili S, Violi F: Atorvastatin inhibits oxidative stress via adiponectin-mediated

of 42Diabetes

23

NADPH oxidase down-regulation in hypercholesterolemic patients. Atherosclerosis

2010;213:225-234

24. Yuzefovych LV, LeDoux SP, Wilson GL, Rachek LI: Mitochondrial DNA damage via

augmented oxidative stress regulates endoplasmic reticulum stress and autophagy:

crosstalk, links and signaling. PLoS One 2013;8:e83349

25. Klionsky DJ, et al: Guidelines for the use and interpretation of assays for monitoring

autophagy. Autophagy 2012;8:445-544

26. Lin Z, Wu F, Lin S, Pan X, Jin L, Lu T, Shi L, Wang Y, Xu A, Li X: Adiponectin

protects against acetaminophen-induced mitochondrial dysfunction and acute liver injury

by promoting autophagy in mice. J Hepatol 2014;S0168-8278:00380-00388

27. Qi GM, Jia LX, Li YL, Li HH, Du J: Adiponectin Suppresses Angiotensin II-Induced

Inflammation and Cardiac Fibrosis through Activation of Macrophage Autophagy.

Endocrinology 2014;155:2254-2265

28. Wu JJ, Quijano C, Chen E, Liu H, Cao L, Fergusson MM, Rovira, II, Gutkind S,

Daniels MP, Komatsu M, Finkel T: Mitochondrial dysfunction and oxidative stress

mediate the physiological impairment induced by the disruption of autophagy. Aging

(Albany NY) 2009;1:425-437

29. Wen H, Gris D, Lei Y, Jha S, Zhang L, Huang MT, Brickey WJ, Ting JP: Fatty acid-

induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nat

Immunol 2011;12:408-415

30. Castets P, Ruegg MA: MTORC1 determines autophagy through ULK1 regulation in

skeletal muscle. Autophagy 2013;9

31. Castets P, Lin S, Rion N, Di Fulvio S, Romanino K, Guridi M, Frank S, Tintignac LA,

Sinnreich M, Ruegg MA: Sustained activation of mTORC1 in skeletal muscle inhibits

constitutive and starvation-induced autophagy and causes a severe, late-onset

myopathy. Cell Metab 2013;17:731-744

32. Nepal S, Park PH: Activation of autophagy by globular adiponectin attenuates

ethanol-induced apoptosis in HepG2 cells: involvement of AMPK/FoxO3A axis. Biochim

Biophys Acta 2013;1833:2111-2125

33. Lin Z, Tian H, Lam KS, Lin S, Hoo RC, Konishi M, Itoh N, Wang Y, Bornstein SR, Xu

A, Li X: Adiponectin mediates the metabolic effects of FGF21 on glucose homeostasis

and insulin sensitivity in mice. Cell Metab 2013;17:779-789

34. Holland WL, Adams AC, Brozinick JT, Bui HH, Miyauchi Y, Kusminski CM, Bauer

SM, Wade M, Singhal E, Cheng CC, Volk K, Kuo MS, Gordillo R, Kharitonenkov A,

of 42 Diabetes

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Scherer PE: An FGF21-adiponectin-ceramide axis controls energy expenditure and

insulin action in mice. Cell Metab 2013;17:790-797

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Figure Legends

Figure 1. Metabolic characterization of wild type and adiponectin knockout mice ±

adiponectin administration

Wild type (WT) or adiponectin knockout (KO) mice were fed either regular chow (Chow)

or 60% high fat diet (HFD) at the age of 6wks for a period of 2, 4 or 6 wks. Mice were

weighted and experiments performed, or serum samples collected for analysis, after 5-

6hrs fasting. Skeletal muscle insulin signaling was assessed 15 min after a bolus

injection of insulin (4 U/kg body weight) via tail vein. During intraperitoneal glucose

tolerance test (IPGTT), blood samples were collected and glucose level determined 15,

30, 60, 90 mins after a bolus intraperitoneal injection of glucose. A. IPGTT-area under

curve (AUC); B. Fasting insulin level (before IPGTT, ng/ml); C. Fasting glucose level

(before IPGTT, mM); D. Homeostatic model assessment for insulin resistant (HOMA-IR)

were calculated using the formula: (fasting glucose(mM)*fasting insulin(mU/L))/22.5; E.

Quantitative analysis of Western blot to determine insulin stimulated p-Akt(S473) and p-

IRS1(Y612) in skeletal muscle, p-Akt data were corrected by total Akt2 and pIRS1 data

were corrected by GAPDH. Data represent mean ± SEM; n=5-11. * indicates significant

difference between HFD and chow in WT animal; # indicates significant difference

between HFD and chow in AdKO; $ indicates significant difference between WT and

AdKO mice; & significant difference during time course within one genotype. *,#,$,&

P<0.05; **,##,$$,&&; P<0.01; ***,###,$$$,&&&; P<0.001.

Figure 2. Hyperinsulinemic-euglycemic clamp analysis

Ad-KO mice were fed either regular chow (Chow) or 60% high fat diet (HFD) at the age

of 6 wks for the period of 2 wks. Jugular vein and carotid artery catheters were

embedded into animals 4 days before the hyperinsulinemic euglycemic clamp

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procedure, and clamps were performed on animals after 5-6 h starvation. Blood samples

were collected during the clamp procedure and calculations were made based on the

radioactivity readings from serum to represent whole body glucose metabolism. A.

Glucose infusion rate (GIR) (mg/kg/min); B. Glucose appearance rate (Ra:mg/kg/min);

C. Glucose disappearance rate (Rd:mg/kg/min); D. Glycolytic rate (mg/kg/min); E.

Glucose turnover rate (fold change calculated by post (after insulin clamp)/basal (before

insulin clamp)). Data represent mean ± SEM; n=4-5. * significant compare between HFD

and chow animal; # significant difference between before (basal) and after (post) insulin

clamp. *,# P<0.05; **,## P<0.01; ***,### P<0.001.

Figure 3. Analysis of oxidative stress in skeletal muscle


or 60% high fat diet (HFD) at the age of 6wks for the period of 2,4 and 6 wks. Skeletal

muscle samples were collected after 5-6 hrs fasting for subsequent analysis of A. SOD

activity and B. Ratio between reduced glutathione (GSH) over oxidized glutathione

(GSSG). Data represent mean ± SEM; n=4-8. * significant difference between HFD and

chow in WT animal; # significant difference between HFD and chow in KO; $ significant

difference between WT and KO mice. KO mice were fed either regular chow (Chow) or

60% high fat diet (HFD) at the age of 6wks. After 2 wks, mice were treated with either

saline (Chow, 60% HF) or fAd (60% HF + fAd) at dosage of 3ug/g body weight twice a

day via intraperitoneal injection for an additional 1 wk. Skeletal muscle samples were

collected after 5-6 hrs starvation to analyze C. SOD activity; D. TBARS assay. Data

represent mean ± SEM; n=6-7. * significant difference comparing HFD and chow; #

significant difference between saline and adiponectin treated HFD-fed mice. Primary

skeletal muscle cells isolated from C57BL6 mice were cultured with or without

adiponectin (Ad; 5ug/ml) until differentiated into myotubes then treated with 250uM

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palmitate for 2 hrs (E) and 48 hrs (F) followed by analysis of oxidative stress by ROS

production (E) and insulin resistance by glucose uptake (F). Flow cytometry was used to

detect intracellular ROS followed by H2DCFDA staining for 30mins (E). Insulin was used

at submaximal (10nM) and maximal (100nM) concentrations during the final 1 hr before

glucose uptake assay (F; pmol/mg/min). Data represent mean ± SEM; n=4-5. *

significant difference compared to control; # significant difference compared to palmitate

treatment. *,#,$ P<0.05; **,##,$$ P<0.01; ***,###,$$$ P<0.001.

Figure 4. Oxidative stress gene array

Ad-KO mice were fed either regular chow or 60% high fat diet (HFD) at the age of 6wks.

After 2wks, the mice were treated with either saline or adiponectin at a dosage of 3ug/g

body weight twice a day via intraperitoneal injection for an additional 1 wk. Skeletal

muscle samples were collected after 5-6 hrs starvation and mRNA extracted and

analyzed by PCR array. A. Pie chart of global dataset (84 genes) indicating differentially

expressed genes after HFD treatment categorized based on pathways involved in the

regulation of oxidative stress; B. Quantitative analysis of gene expressions that were

most highly altered by HFD and/or adiponectin administration. n=4-5.

Figure 5. Analysis of skeletal muscle autophagy using Western blotting


or 60% high fat diet (HFD) at the age of 6wks for the period of 2,4 and 6 wks. In

additional studies with Ad-KO mice, after 2 wks and 6 wks these mice were treated with

either saline or adiponectin (Ad) at a dosage of 3ug/g body weight twice a day via

intraperitoneal injection for an additional 1 wk and 2 wks, respectively. Skeletal muscle

samples were collected after 5-6 hrs fasting for subsequent analysis by Western blotting.

In WT and KO animals on Chow or HFD for 2, 4 and 6 weeks we examined expression

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of LC3 (A), p62 (C), Beclin1 (E) and FGF21 (G), all corrected for GAPDH content. n=8

and * indicates P<0.05 compared to Chow. We also examined the effect of adiponectin

replenishment after 2 or 6 wks HFD in Ad-KO mice on expression of LC3 (B), p62 (D),

Beclin1 (F) and FGF21 (H). Expression of autophagy-related genes were also

determined by RT-PCR (n=6) and changes in relative expression levels shown as fold

changes in heat map (I). In A-H, n=8 and * indicates P<0.05 compared to Chow and #

indicates P<0.05 comparing HFD + Ad to HFD alone.

Figure 6. Stimulation of autophagy flux by adiponectin in L6 cells.

(A) L6 cells were treated with adiponectin (5ug/ml) for 1hr or 2hrs in the presence or

absence of compound C (10µM, 1hr) then LC3 and phospho-ULK1 (Ser555) analyzed

by Western blotting cell lysates. Representative images and quantitation of n=3-5

experiments (mean ± SEM) are shown. *P>0.05 comparing control versus adiponectin

treatment. (B) Immunofluorescent detection of intracellular endogenous LC3 by confocal

microscopy. Nucleus was identified using DAPI. Representative images for DAPI, LC3

and merged image are shown on left side with higher magnification of single cells on

right side. (C) Analysis of tandem RFP/GFP-LC3 expressing L6 cells showing

representative images from n=3 of the relative GFP and RFP signals, and merged

image. (D) Representative fluorescence images of Magic Red, cathepsin B activity,

detected by confocal microscopy.

Figure 7. Functional significance of altered autophagy on insulin sensitivity

(A) L6 cells stably overexpressing GLUT4-myc were pretreated ± bafilomycin (200nM,

24hr) or chloroquine (100µM, 24hr) and stimulated with insulin (10 or 100 nM, 20 min)

prior to anlaysis of glucose uptake. In B-E, cells were pretreated ± bafilomycin or

chloroquine and stimulated with insulin (10 or 100 nM, 5 min) then cell lysates prepared

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to determine phosphorylation of IRS Y612 (B&C), Akt T308 (B&D) and Akt S473 (B&E).

An autophagy-deficient stable cell line was created using Atg5K130R overexpression

and comparing these cells (Atg5K) versus cells infected with empty vector (EV) shows

that insulin sensitivity (1, 10 and 100 nM, 5 min) was attenuated (F-I). n=3-5 and *

indicates P<0.05 compared to no insulin and # indicates P<0.05 comparing insulin action

in the Atg5K versus EV cells.

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Supplementary Figure 1

Representative Western blots for phosphoAkt (S473) and phosphoIRS (Y612) in WT and

AdKO mice 2,4,6 weeks after chow or HFD. Quantitative analysis of these samples is

shown in figure 1.


Complete dataset for the list of all oxidative stress-related genes tested by PCR array,

categorized by functional gene group (left column) and heat map data showing the fold

change in gene expression in response to HFD (HFD:chow) or Ad (HFD+Ad:HFD). Red

indicates reduced, and green indicates increased gene expression.


Representative Western blots for LC3, p62, Beclin1 and FGF21 in WT and AdKO mice

2,4,6 weeks after chow or HFD. Quantitative analysis of samples shown in figure 5.


Representative Western blots for LC3, p62, Beclin1 and FGF21 in AdKO mice at 2

and/or 6 weeks after HFD feeding ± adiponectin replenishment. Quantitative analysis of

samples shown in figure 5.

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